The complete genome of 2,6-dichlorobenzamide (BAM) degrader Aminobacter sp. MSH1 suggests a polyploid chromosome, phylogenetic reassignment, and functions of plasmids.

Aminobacter sp. MSH1 (CIP 110285) can use the pesticide dichlobenil and its recalcitrant transformation product, 2,6-dichlorobenzamide (BAM), as sole source of carbon, nitrogen, and energy. The concentration of BAM in groundwater often exceeds the threshold limit for drinking water, requiring additional treatment in drinking water treatment plants or closure of the affected abstraction wells. Biological treatment with MSH1 is considered a potential sustainable alternative to remediate BAM-contamination in drinking water production. We present the complete genome of MSH1, which was determined independently in two institutes at Aarhus University and KU Leuven. Divergences were observed between the two genomes, i.e. one of them lacked four plasmids compared to the other. Besides the circular chromosome and the two previously described plasmids involved in BAM catabolism, pBAM1 and pBAM2, the genome of MSH1 contained two megaplasmids and three smaller plasmids. The MSH1 substrain from KU Leuven showed a reduced genome lacking a megaplasmid and three smaller plasmids and was designated substrain MK1, whereas the Aarhus variant with all plasmids was designated substrain DK1. A plasmid stability experiment indicate that substrain DK1 may have a polyploid chromosome when growing in R2B medium with more chromosomes than plasmids per cell. Finally, strain MSH1 is reassigned as Aminobacter niigataensis MSH1.

Discovery of N-((3S,4S)-4-(3,4-Difluorophenyl)piperidin-3-yl)-2-fluoro-4-(1-methyl-1H-pyrazol-5-yl)benzamide (Hu7691), a Potent and Selective Akt Inhibitor That Enables Decrease of Cutaneous Toxicity.

Rash is one of the primary dose-limiting toxicities of Akt (protein kinase B) inhibitors in clinical trials. Here, we demonstrate the inhibition of Akt2 isozyme may be a driver for keratinocyte apoptosis, which promotes us to search for new selective Akt inhibitors with an improved cutaneous safety property. According to our previous research, compound 2 is selected for further optimization for overcoming the disadvantages of compound 1, including high Akt2 inhibition and high toxicity against HaCaT keratinocytes. The dihedral angle-based design and molecular dynamics simulation lead to the identification of Hu7691 (B5) that achieves a 24-fold selectivity between Akt1 and Akt2. Hu7691 exhibits low activity in inducing HaCaT apoptosis, promising kinase selectivity, and excellent anticancer cell proliferation potencies. Based on the superior results of safety property, pharmacokinetic profile, and in vivo efficacy, the National Medical Products Administration (NMPA) approved the investigational new drug (IND) application of Hu7691.

Managing toxicities of Bruton tyrosine kinase inhibitors.

Inhibition of Bruton's tyrosine kinase (BTK) has revolutionized the treatment landscape for patients with chronic lymphocytic leukemia (CLL). By targeting this critical kinase in proximal B-cell receptor signaling, BTK inhibitors (BTKis) impair cell proliferation, migration, and activation of NF-κB. Clinically, because indefinite inhibition is a mainstay of therapy, there is an extended period of exposure in which adverse effects can develop. Given the impressive efficacy and activity of BTKis in the treatment of patients with CLL, appropriate management of treatment-emergent adverse events (AEs) is of paramount importance. Here we review the BTKi landscape and present the available toxicity and safety data for each agent. The long-term toxicity profile of ibrutinib, a first-in-class inhibitor, is well characterized and includes a clinically significant incidence of cardiac arrhythmias, bleeding, infection, diarrhea, arthralgias, and hypertension. Acalabrutinib, the initial second-generation BTKi to earn approval from the US Food and Drug Administration, demonstrates improved kinase selectivity for BTK, with commonly observed adverse reactions including infection, headache, and diarrhea. Mediated by both on-target inhibition of BTK and variable off-target inhibition of other kinases including interleukin-2-inducible T-cell kinase (ITK), tyrosine-protein kinase (TEC), and endothelial growth factor receptor (EGFR), the toxicity profile of BTKis is closely linked to their pattern of kinase binding. Other emerging BTKis include second-generation agents with variable degrees of kinase selectivity and third-generation agents that exhibit reversible noncovalent binding to BTK. We also highlight critical considerations for the prevention and monitoring of AEs and offer practical management strategies for treatment-emergent toxicities.

Phase 1 study of the HSP90 inhibitor onalespib in combination with AT7519, a pan-CDK inhibitor, in patients with advanced solid tumors.

PURPOSE: We conducted a phase 1 trial of the HSP90 inhibitor onalespib in combination with the CDK inhibitor AT7519, in patients with advanced solid tumors to determine the safety profile and maximally tolerated dose, pharmacokinetics, preliminary antitumor activity, and to assess the pharmacodynamic (PD) effects on HSP70 expression in patient-derived PBMCs and plasma. METHODS: This study followed a 3 + 3 trial design with 1 week of intravenous (IV) onalespib alone, followed by onalespib/AT7519 (IV) on days 1, 4, 8, and 11 of a 21-days cycle. PK and PD samples were collected at baseline, after onalespib alone, and following combination therapy. RESULTS: Twenty-eight patients were treated with the demonstration of downstream target engagement of HSP70 expression in plasma and PBMCs. The maximally tolerated dose was onalespib 80 mg/m2 IV + AT7519 21 mg/m2 IV. Most common drug-related adverse events included Grade 1/2 diarrhea (79%), fatigue (54%), mucositis (57%), nausea (46%), and vomiting (50%). Partial responses were seen in a palate adenocarcinoma and Sertoli-Leydig tumor; a colorectal and an endometrial cancer patient both remained on study for ten cycles with stable disease as the best response. There were no clinically relevant PK interactions for either drug. CONCLUSIONS: Combined onalespib and AT7519 is tolerable, though below monotherapy RP2D. Promising preliminary clinical activity was seen. Further benefit may be seen with the incorporation of molecular signature pre-selection. Further biomarker development will require the assessment of the on-target impact on relevant client proteins in tumor tissue.

In Vitro Assessment of the Genotoxic Hazard of Novel Hydroxamic Acid- and Benzamide-Type Histone Deacetylase Inhibitors (HDACi).

Histone deacetylase inhibitors (HDACi) are already approved for the therapy of leukemias. Since they are also emerging candidate compounds for the treatment of non-malignant diseases, HDACi with a wide therapeutic window and low hazard potential are desirable. Here, we investigated a panel of 12 novel hydroxamic acid- and benzamide-type HDACi employing non-malignant V79 hamster cells as toxicology guideline-conform in vitro model. HDACi causing a ≥10-fold preferential cytotoxicity in malignant neuroblastoma over non-malignant V79 cells were selected for further genotoxic hazard analysis, including vorinostat and entinostat for control. All HDACi selected, (i.e., KSK64, TOK77, DDK137 and MPK77) were clastogenic and evoked DNA strand breaks in non-malignant V79 cells as demonstrated by micronucleus and comet assays, histone H2AX foci formation analyses (γH2AX), DNA damage response (DDR) assays as well as employing DNA double-strand break (DSB) repair-defective VC8 hamster cells. Genetic instability induced by hydroxamic acid-type HDACi seems to be independent of bulky DNA adduct formation as concluded from the analysis of nucleotide excision repair (NER) deficient mutants. Summarizing, KSK64 revealed the highest genotoxic hazard and DDR stimulating potential, while TOK77 and MPK77 showed the lowest DNA damaging capacity. Therefore, these compounds are suggested as the most promising novel candidate HDACi for subsequent pre-clinical in vivo studies.

In vitro and in vivo effects of flubendiamide and copper on cyto-genotoxicity, oxidative stress and spleen histology of rats and its modulation by resveratrol, catechin, curcumin and α-tocopherol.

BACKGROUND: Living organisms are frequently exposed to more than one xenobiotic at a time either by ingestion of contaminated food/fodder or due to house-hold practices, occupational hazards or through environment. These xenobiotics interact individually or in combination with biological systems and act as carcinogen or produce other toxic effects including reproductive and degenerative diseases. Present study was aimed to investigate the cyto-genotoxic effects of flubendiamide and copper and ameliorative potential of certain natural phyotconstituent antioxidants. METHOD: In vitro cytogenotoxic effects were evaluated by employing battery of assays including Propidium iodide staining, Tunel assay, Micronuclei, DNA fragmentation and Comet assay on isolated splenocytes and their prevention by resveratrol (5 and 10 µM), catechin (10 and 20 µM), curcumin (5 and 10 µM) and α-tocopherol (5, 10 and 20 µM). In vivo study was also undertaken daily oral administration of flubendiamide (200 mg/kg) or copper (33 mg/kg) and both these in combination, and also all these concurrently with of α-tocopherol to Wistar rats for 90 days. RESULTS: Flubendiamide and copper produced concentration-dependent cytotoxic effects on splenocytes and at median lethal concentrations, flubendiamide (40 µM) and copper (40 µM) respectively produced 71 and 81% nonviable cells, higher number of Tunel+ve apoptotic cells, 7.86 and 9.16% micronucleus and 22.90 and 29.59 comets/100 cells and DNA fragmentation. In vivo study revealed significant (P < 0.05) increase in level of lipid peroxidation (LPO) and decrease in glutathione peroxidase (GPx), glutathione-S-transferase (GST) and superoxide dismutase (SOD) activities in groups exposed to flubendiamide or copper alone or both these in combination. Histopathological examination of rat spleens revealed depletion of lymphoid tissue, separation of splenocytes and rarification in splenic parenchyma of xenobiotic(s) treated groups. CONCLUSION: Flubendiamide and copper induce oxidative stress and produce cytogenotoxic effects along with histoarchitectural changes in spleen. All four tested natural antioxidants (resveratrol, catechin, curcumin and α-tocopherol) reduced flubendiamide and copper-induced cytotoxic effects in rat splenocytes. Rat splenocytes are very sensitive to flubendiamide and copper-induced cytogenotoxicity, therefore, these can be effectively employed for screening of compounds for their cytogenotoxic potential. α-tocopherol was effective in restoring alterations in oxidative stress biomarkers and preventing histoarchitectural lesions in spleen.

Triflumuron induces genotoxicity in both mice bone marrow cells and human Colon cancer cell line.

Triflumuron (TFM) is an insect growth regulator (IGR), an insecticide commonly used over the world. It is known for its several toxic manifestations, such as reprotoxicity, immunotoxicity and hematotoxicity, which could affect public health. However, studies that reveal its toxic effects on mammalians are limited. To reach this purpose, our study aimed to elucidate the eventual genotoxic effects of TFM in mice bone marrow cells and in HCT 116 cells after a short term exposition. TFM was administered intraperitoneally to Balb/C male mice at doses of 250, 350 and 500 mg/kg bw for 24 h. Genotoxicity was monitored in bone marrow cells using the comet test, the micronucleus test and the chromosome aberration assay. Our results showed that TFM induced DNA damages in a dose-dependent manner. This genotoxicity was confirmed also in vitro on human intestinal cells HCT 116 using the comet test. It was then asked whether this genotoxicity induced by TFM could be due to an oxidative stress. Thus, we found that TFM significantly decreased HCT 116 cell viability. In addition, it induced the generation of reactive oxygen species (ROS) followed by lipid peroxidation as revealed by the increase in the malondialdehyde (MDA) levels. Similarly, the activation of the antioxidant enzymes (catalase and superoxide dismutase) was also observed. Our results indicated that, in our experimental conditions, TFM had a genotoxic effect on bone morrow cells and in HCT 116 cells. Moreover, we demonstrated that this genotoxicity passes through an oxidative stress.

Protective effects of fennel essential oil against oxidative stress and genotoxicity induced by the insecticide triflumuron in human colon carcinoma cells.

The increased use of pesticides is the origin of multiple damages to the environment and to humans; thus, the search for new strategies to reduce or even protect the toxic effects caused by these synthetic products became a necessity. In this context, our study attempted to evaluate the protective effects of fennel essential oil (FEO), the main essential oil extracted from Faeniculum vulgare Mill., a plant with aromatic, flavorful, and medicinal uses, against toxicity induced by an insecticide-triflumuron (TFM)-in human carcinoma cells (HCT116). Our methodological approach consists of the cytotoxicity assay starting with the cell viability test, the ROS generation, the malondialdehyde (MDA) production, the DNA fragmentation, and the measurement of some antioxidant enzymes activities such as catalase (CAT) and superoxide dismutase (SOD). Also, we measured the mitochondrial transmembrane potential. The outcome of the current study showed clearly that after 2 h of HCT 116 cell pretreatment with FEO, there were increase in cell viability, reduction in ROS generation, and modulation in CAT and SOD activities induced by TFM. In the same manner, significant decreases in MDA levels were found. Mainly, the results indicated a perceptible decrease in DNA damages and a significant reduction in the mitochondrial membrane potential loss. Our work demonstrates that FEO can be an important protector against toxic effects induced by TFM in HCT 116 cells.

Genotoxic action of Luna Experience-SC 400 fungicide on rat bone marrow.

Background: Fungicides describe all chemicals used to control fungi that infect plants. Luna Experience SC-400 is a new line of fungicide that consist of Fluopyram and Tebuconazole. Objective: In this study, We investigated the genotoxicty and cytotoxicty of Luna Experience-SC 400 using comet assay, micronucleus test and polychromatic erythrocytes number in rat bone marrow. The present study is the first report indicating the effects of genotoxic and cytotoxic of Luna experience SC-400 on rat bone marrow cells. Material and Methods: We used three different doses (5mg/kg, 10mg/kg, 20mg/kg) of Luna Experience SC 400 at 48 h intervals during 30 days by gavage in rats.Genotoxicity was evaluated using comet assay and micronucleus test and cytotoxicity was measured the PCE/NCE rate in rat bone marrow. Results: Based on these experimental results, we report that Luna Experience-SC 400 fungicide presents genotoxic and cytotoxic potential on rat bone marrow. There is a significant difference between negative control group and all the doses of Luna Experience-SC 400 (p < 0.05) for comet assay and micronucleus. Even moderate and high doses of fungicides seem to have reached the values of almost positive control group for Genetic Damage Index (GDI) and Damaged Cell Percentage (DCP). In this study, we also investigated the PCE/NCE rate. Fungicide caused a decrease in the level of significant in the PCE/NCE ratio (p < 0.05). Conclusion: Our in vivo study suggests that the gavage exposure to Luna experience SC 400 used in the present investigation may be genotoxic and cytotoxic in rat bone marrow in view of these findings. Because this findings is first report represented in the pesticide biology, it is important to carry out more investigations using various cytogenetic tests under different experimental conditions to definitively resolve the the possible genotoxic and cytotoxic risk associated with new generation pesticides-fungicides.

Thioether-based 2-aminobenzamide derivatives: Novel HDAC inhibitors with potent in vitro and in vivo antitumor activity.

Previously, we focused on a series of 2-aminobenzamide-based histone deacetylase (HDAC) inhibitors, compound 9 of which displayed potent HDAC inhibitory activity against HDAC1 and HDAC2, and moderate anti-proliferative activity against several cancer cell lines. In the current study, we have designed and synthesized a series of novel HDAC inhibitors based on thioether moiety with 9 as a lead compound. Representative compounds12 g and 12 h showed apparently potent anti-proliferative activities against five solid cancer cell lines: A549, HCT116, Hela, A375 and SMMC7721, and low cytotoxicity against NIH 3T3 normal cells. Especially, 12 g and 12 h also revealed potent HDAC inhibitory activity against HDAC1, 2 and 3. In addition, the two compounds could arrest cell cycle in G2/M phase and promote cell apoptosis. Moreover, they showed extended inhibition of colony formation and effectively blocked cell migration towards A549 cancer cells. Furthermore, 12 g and 12 h possessed better pharmacokinetic properties than the lead compound 9. Benefiting from these results, we also explored 12 g and 12 h in the A549 xenografts model for in vivo antitumor activity. The in vivo experiment indicated that 12 g and 12 h could evidently augment antitumor activity (TGI = 56.9% and 62.7% respectively).

Benzoic acid-derived nitrones: A new class of potential acetylcholinesterase inhibitors and neuroprotective agents.

The discovery of new chemical entities endowed with potent and selective acetylcholinesterase (AChE) and/or butyrylcholinesterase (BChE) inhibitory activity is still a relevant subject for Alzheimer's disease therapy. Therefore, a small library of benzoic based amide nitrones (compounds 24 to 42) was synthesized and screened toward cholinesterase enzymes. SAR studies showed that the tert-butyl moiety is the most favourable nitrone pattern. In general, tert-butyl derivatives effectively inhibited AChE, being compound 33 the most potent (IC50 = 8.3 ±â€¯0.3 µM; Ki 5.2 µM). The data pointed to a non-competitive inhibition mechanism of action, which was also observed for the standard donepezil. None of compounds showed BChE inhibitory activity. Molecular modelling studies provided insights into the enzyme-inhibitor interactions and rationalised the experimental data, confirming that the binding mode of nitrones 33 and 38 towards AChE has the most favourable binding free energy. The tert-butylnitrones 33 and 38 were not cytotoxic on different cell lines (SH-SY5Y and HepG2). Moreover, compound 33 was able to prevent t-BHP-induced oxidative stress in SH-SY5Y differentiated cells. Due to its AChE selectivity and promising cytoprotective properties, as well as its appropriate drug-like profile pointing toward blood-brain barrier permeability, compound 33 is proposed as a valid lead for a further optimization step.

Optimizing Platelet GPVI Inhibition versus Haemostatic Impairment by the Btk Inhibitors Ibrutinib, Acalabrutinib, ONO/GS-4059, BGB-3111 and Evobrutinib.

Ibrutinib and acalabrutinib are approved for B cell malignancies and novel Bruton's tyrosine kinase (Btk) inhibitors undergo clinical testing also in B cell-driven autoimmune disorders. Btk in platelets mediates platelet activation via glycoprotein (GP) VI, which is crucial for atherosclerotic plaque-induced platelet thrombus formation. This can be selectively inhibited by Btk inhibitors. Since patients on second-generation Btk inhibitors apparently show less bleeding than patients on ibrutinib, we compared the effects of ibrutinib and four novel irreversible Btk inhibitors on GPVI-dependent platelet aggregation in blood and in vitro bleeding time. Low concentrations of collagen which induced the same low degree of GPVI-mediated platelet aggregation as atherosclerotic plaque material were applied. IC50 values for collagen (0.2-0.5 µg/mL)-induced platelet aggregation after 15-minute pre-incubation were: ibrutinib 0.12 µM, BGB-3111 0.51 µM, acalabrutinib 1.21 µM, ONO/GS-4059 1.20 µM and evobrutinib 5.84 µM. Peak venous plasma concentrations of ibrutinib (0.5 µM), acalabrutinib (2 µM) and ONO/GS-4059 (2 µM) measured after anti-proliferative dosage inhibited collagen-induced platelet aggregation, but did not increase PFA-200 closure time on collagen/epinephrine. Closure times were moderately increased by 2- to 2.5-fold higher concentrations of these inhibitors, but not by BGB-3111 (1 µM) and evobrutinib (10 µM). Prolonging platelet drug exposure to 60 minutes lowered IC50 values of any Btk inhibitor for GPVI-mediated aggregation by several fold, and 5- to 10-fold below anti-proliferative therapeutic drug plasma levels. In conclusion, low blood concentrations of ibrutinib and the novel Btk inhibitors suffice for GPVI selective platelet inhibition relevant for atherothrombosis but do not impair primary haemostasis.

Differential expression of cyclin-dependent kinases in the adult human retina in relation to CDK inhibitor retinotoxicity.

Cyclin-dependent kinases (CDKs) are a family of kinases associated predominantly with cell cycle control, making CDK inhibitors interesting candidates for anti-cancer therapeutics. However, retinal toxicity (loss of photoreceptors) has been associated with CDK inhibitors, including the pan-CDK inhibitor AG-012896. The purpose of this research was to use a novel planar sectioning technique to determine CDK expression profiles in the ex vivo human retina with the aim of identifying isoforms responsible for CDK retinotoxicity. Four CDK isoforms (CDK11, 16, 17 and 18) were selected as a result of IC50 data comparing neurotoxic (AG-012986 and NVP-1) and non-neurotoxic (dinaciclib and NVP-2) CDK inhibitors, with IC50s at CDK11 showing a clear difference between the neurotoxic and non-neurotoxic drugs. CDK11 was maximally expressed in the photoreceptor layer, whereas CDK16, 17 and 18 showed maximal expression in the inner nuclear layer. CDK5 (an isoform associated with retinal homeostasis) was maximally expressed in the retinal ganglion cell layer. Apart from CDK18, each isoform showed expression in the photoreceptor layer. The human Müller cell line MIO-M1 expressed CDK5, 11, 16 and 17 and AG-01298 (0.02-60 µM) caused a dose-dependent increase in MIO-M1 cell death. In conclusion, CDK11 appears the most likely candidate for mediation of photoreceptor toxicity. RNA profiling can be used to determine the distribution of genes of interest in relation to retinal toxicity in the human retina.

Comparison of Chlorantraniliprole and Flubendiamide Activity Toward Wild-Type and Malignant Hyperthermia-Susceptible Ryanodine Receptors and Heat Stress Intolerance.

Chlorantraniliprole (CP) and flubendiamide (FD) are widely used in agriculture globally to control lepidopteran pests. Both insecticides target ryanodine receptors (RyRs) and promote Ca2+ leak from sarcoplasmic reticulum (SR) within insect skeletal muscle yet are purportedly devoid of activity toward mammalian RyR1 and muscle. RyRs are ion channels that regulate intracellular Ca2+ release from SR during physiological excitation-contraction coupling. Mutations in RYR1 genes confer malignant hyperthermia susceptibility (MHS), a potentially lethal pharmacogenetic disorder in humans and animals. Compared with vehicle control, CP (10 µM) triggers a 65-fold higher rate of Ca2+ efflux from Ca2+-loaded mammalian WT-RyR1 SR vesicles, whereas FD (10 µM) produces negligible influence on Ca2+ leak. We, therefore, compared whether CP or FD differentially influence patterns of high-affinity [3H]ryanodine ([3H]Ry) binding to RyR1 isolated from muscle SR membranes prepared from adult C57BL/6J mice expressing WT, homozygous C-terminal MHS mutation T4826I, or heterozygous N-terminal MHS mutation R163C. Basal [3H]Ry binding differed among genotypes with rank order T4826I ≫R163C∼WT, regardless of [Ca2+] in the assay medium. Both CP and FD (0.01-100 µM) elicited concentration-dependent increase in [3H]Ry binding, although CP showed greater efficacy regardless of genotype or [Ca2+]. Exposure to CP (500 mg/kg; p.o) failed to shift intolerance to heat stress (38°C) characteristic of R163C and T4826I MHS mice, nor cause lethality in WT mice. Although nM-µM of either diamide is capable of differentially altering WT and MHS RyR1 conformation in vitro, human RyR1 mutations within putative diamide N- and C-terminal interaction domains do not alter heat stress intolerance (HSI) in vivo.

Novel 4-(3-phenylpropionamido), 4-(2-phenoxyacetamido) and 4-(cinnamamido) substituted benzamides bearing the pyrazole or indazole nucleus: synthesis, biological evaluation and mechanism of action.

Based on some common structural features of known compounds interfering with p53 pathways and our previously synthesized benzamides, we synthesized new ethyl 5-(4-substituted benzamido)-1-phenyl-1H-pyrazole-4-carboxylates 26a-c, ethyl 5-(4-substituted benzamido)-1-(pyridin-2-yl)-1H-pyrazole-4-carboxylates 27a-c and N-(1H-indazol-6-yl)-4-substituted benzamides 31a,b bearing in the 4 position of the benzamido moiety the 2-phenylpropanamido or 2-phenoxyacetamido or cinnamamido groups. A preliminary test to evaluate the antiproliferative activity against human lung carcinoma H292 cells highlighted how compound 26c showed the best activity. This last was therefore selected for further studies with the aim to find the mechanism of action. Compound 26c induces intrinsic apoptotic pathway by activating p53 and is also able to activate TRAIL-inducing death pathway by promoting increase of DR4 and DR5 death receptors, downregulation of c-FLIPL and caspase-8 activation.

A novel ASBT inhibitor, IMB17-15, repressed nonalcoholic fatty liver disease development in high-fat diet-fed Syrian golden hamsters.

The manipulation of bile acid (BA) homeostasis by blocking the ileal apical Na+-dependent bile salt transporter (ASBT/SLC10A2) may have therapeutic effects in nonalcoholic fatty liver disease. We developed a novel ASBT inhibitor, an N-(3,4-o-dichlorophenyl)-2-(3-trifluoromethoxy) benzamide derivative referred to as IMB17-15, and investigated its therapeutic effects and the molecular mechanisms underlying the effects. Syrian golden hamsters were challenged with high-fat diet (HFD) to induce NAFLD and were subsequently administered 400 mg/kg IMB17-15 by gavage daily for 21 days. Serum, liver, and fecal samples were collected for further analysis. Plasma concentration-time profiles of IMB17-15 were also constructed. The human hepatocyte cell line HL-7702 was treated with Oleic acid (OA) with or without IMB17-15. Western blotting and real-time PCR were used to study the molecular mechanisms of IMB17-15. We found that IMB17-15 inhibited ASBT and subsequently suppressed ileal farnesoid X receptor (FXR) and FXR-activated fibroblast growth factor15/19 (FGF15/19) expression, which reduced the hepatic phosphorylated extracellular regulated protein kinase (ERK) and c-Jun N-terminal kinase (JNK) levels and upregulated the cholesterol 7α-hydroxylase (CYP7A1) activity. Additionally, IMB17-15 stimulated adenosine monophosphate (AMP)-activated protein kinase (AMPKα) phosphorylation and enhanced peroxisome proliferator activated receptor α (PPARα) expression and thus promoted triglyceride (TG) oxidation and high-density lipoprotein cholesterol (HDL-c) metabolism through an ASBT-independent mechanism. In conclusion, a novel ASBT inhibitor known as IMB17-15 protected hamsters against HFD-induced NFALD by manipulating BA and lipid homeostasis. IMB17-15 also reduced lipid deposition in human hepatic cell lines, indicating that it may be useful as a therapy for NAFLD patients.

Alda-1, an aldehyde dehydrogenase-2 agonist, causes deterioration in renal functions following ischemia-reperfusion injury due to crystalline nephropathy.

Renal ischemia-reperfusion injury (IRI) induces the production of aldehydes which are detoxified by aldehyde dehydrogenases (ALDHs). Alda-1 is a selective ALDH2 agonist and its protective effect was demonstrated in several conditions. The effect of Alda-1 on the kidney or on renal IRI was not investigated. We investigated the effect of Alda-1 on the renal dysfunction following IRI. Wistar rats underwent left IRI for 40 min. Group-Alda (n = 11) received Alda-1 starting 24 h before IRI and continued for 7 days thereafter when renal functions were measured. Group-Vx (n = 11) underwent similar protocol but received the dissolvent. Alda-1 did not affect renal blood flow or glomerular filtration rate in the left ischemic kidney in Group-Alda compared to Group-Vx (3.05 ± 0.50 vs. 3.53 ± 0.70, and 0.40 ± 0.06 vs. 0.51 ± 0.08, respectively, p > .05 for both). However, left renal fractional sodium excretion was higher in Group-Alda (2.80 ± 0.43 vs. 1.37 ± 0.36, p = .02). Alda-1 also adversely affected the gene expressions of kidney injury molecule-1 and neutrophil gelatinase-associated lipocalin (217 ± 38 vs. 99 ± 13 and 49 ± 13 vs. 20 ± 5, respectively, p < .05 for both) and the alterations in tumor necrosis factor-α, transforming growth factor-ß1, plasminogen activator inhibitor-1, fibronectin 1 and p53 (4.4 ± 0.9 vs. 2.1 ± 0.3, 1.5 ± 0.1 vs. 1.1 ± 0.1, 30.0 ± 2.7 vs. 11.7 ± 2.3, 3.6 ± 0.4 vs. 2.1 ± 0.2 and 1.3 ± 0.1 vs. 0.9 ± 0.07, respectively, p ≤ .05 for all). This was associated with intratubular crystal deposition suggestive of crystalline nephropathy. Alda-1 exacerbated the IRI-induced renal tubular dysfunction and alterations in markers of acute kidney injury, biomarkers of inflammation, fibrosis and apoptosis and this was associated with intratubular crystal deposition suggestive of crystalline nephropathy.

Verification of a false positive in a two-year rat carcinogenicity study using dual control groups.

There is sometimes controversy over whether or not statistically significant responses produced in carcinogenicity studies have biologically significance. Ambiguous results from our previous two-year oral carcinogenicity study on acotiamide hydrochloride hydrate (acotiamide-HH), a prokinetic drug for functional dyspepsia, in rats made it unclear whether the drug may exhibit uterine carcinogenicity. To check this finding, we performed a second long-term carcinogenicity study using two identical control groups to more accurately evaluate uterine carcinogenesis by considering the incidence of spontaneous neoplasms. Female Fischer 344 rats were divided into three groups: the two control groups (control 1 and 2) were administered vehicle (0.5% w/v methylcellulose) and the acotiamide-HH-treated group was administered 2,000 mg/kg/day of acotiamide-HH by oral gavage for two years. Among all groups, the incidence of endometrial adenocarcinoma (EmA) was highest in the control 2 group, followed by the acotiamide-HH-treated group and the control 1 group. Moreover, acotiamide-HH did not affect the incidence of precursor lesions of EmA. In cases where an ambiguous difference is observed, the use of two control groups allows for a more informed interpretation of the findings in the drug-treated groups. The outcomes in this study strongly support the hypothesis that the increase in EmA in rats treated with acotiamide-HH in our previous study is unrelated to administration of the drug.

Synthesis, Structural Characterization, and Antiangiogenic Activity of Polyfluorinated Benzamides.

The introduction of fluorine into bioactive molecules is a matter of importance in medicinal chemistry. In this study, representatives of various chemical entities of fluoroaromatic compounds were synthesized. Depending on the reaction conditions, either tetrafluorophthalimides or ammonium tetrafluorophthalamates are accessible from tetrafluorophthalic anhydride and primary amines. Tetrafluorophthalamic acids undergo thermal decarboxylation to yield tetrafluorobenzamides. These could be successfully converted upon treatment with primary amines, in the course of an aromatic nucleophilic substitution, to 2,3,5-trifluorobenzamides with respective amino substituents at the 4-position. The five structure types were characterized by means of spectroscopic and crystallographic methods. The synthesized compounds were evaluated as inhibitors of angiogenesis by measuring microvessel outgrowth in a rat aortic ring assay. The biological activity was maintained throughout these different polyfluorinated chemotypes.

Marbostat-100 Defines a New Class of Potent and Selective Antiinflammatory and Antirheumatic Histone Deacetylase 6 Inhibitors.

Epigenetic modifiers of the histone deacetylase (HDAC) family contribute to autoimmunity, cancer, HIV infection, inflammation, and neurodegeneration. Hence, histone deacetylase inhibitors (HDACi), which alter protein acetylation, gene expression patterns, and cell fate decisions, represent promising new drugs for the therapy of these diseases. Whereas pan-HDACi inhibit all 11 Zn2+-dependent histone deacetylases (HDACs) and cause a broad spectrum of side effects, specific inhibitors of histone deacetylase 6 (HDAC6i) are supposed to have less side effects. We present the synthesis and biological evaluation of Marbostats, novel HDAC6i that contain the hydroxamic acid moiety linked to tetrahydro-ß-carboline derivatives. Our lead compound Marbostat-100 is a more potent and more selective HDAC6i than previously established well-characterized compounds in vitro as well as in cells. Moreover, Marbostat-100 is well tolerated by mice and effective against collagen type II induced arthritis. Thus, Marbostat-100 represents a most selective known HDAC6i and the possibility for clinical evaluation of a HDAC isoform-specific drug.